WO2023154984A1 - Polythérapies - Google Patents

Polythérapies Download PDF

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WO2023154984A1
WO2023154984A1 PCT/AU2023/050108 AU2023050108W WO2023154984A1 WO 2023154984 A1 WO2023154984 A1 WO 2023154984A1 AU 2023050108 W AU2023050108 W AU 2023050108W WO 2023154984 A1 WO2023154984 A1 WO 2023154984A1
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alkyl
compound
alkenyl
tumour
alkynyl
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PCT/AU2023/050108
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English (en)
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Paul Warren Reddell
Peter Gordon Parsons
Glen Mathew BOYLE
Jason Kingsley CULLEN
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QBiotics Pty Ltd
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Priority claimed from AU2022900340A external-priority patent/AU2022900340A0/en
Application filed by QBiotics Pty Ltd filed Critical QBiotics Pty Ltd
Publication of WO2023154984A1 publication Critical patent/WO2023154984A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/336—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having three-membered rings, e.g. oxirane, fumagillin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/10—X-ray therapy; Gamma-ray therapy; Particle-irradiation therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/10—X-ray therapy; Gamma-ray therapy; Particle-irradiation therapy
    • A61N2005/1092—Details
    • A61N2005/1098—Enhancing the effect of the particle by an injected agent or implanted device
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/10—X-ray therapy; Gamma-ray therapy; Particle-irradiation therapy
    • A61N5/1001—X-ray therapy; Gamma-ray therapy; Particle-irradiation therapy using radiation sources introduced into or applied onto the body; brachytherapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/10—X-ray therapy; Gamma-ray therapy; Particle-irradiation therapy
    • A61N5/1077—Beam delivery systems

Definitions

  • Combination Therapies The present application claims priority from Australian Provisional Patent Application No. 2022900340 filed 17 February 2022, the entirety of which is incorporated herein by reference.
  • Field The present invention relates to combination therapies comprising an epoxytigliane compound and a cancer chemotherapeutic agent or irradiation.
  • Pharmaceutical compositions and kits containing epoxytigliane compounds and the cancer chemotherapeutic agents are also described.
  • Background The treatment of cancer has many options including chemotherapeutic agents, irradiation, surgery and immune stimulating agents.
  • Some therapies are developed for particular types of cancer or those defined by particular biological markers. Known therapies often have significant side effects and may not be suitable for some patients.
  • tumours are inoperable and some patients have co-morbidities that reduce the effectiveness of a chemotherapeutic agent or prevent the use of particular therapeutic agents because of side effects.
  • One option for reducing side effects, expanding the therapies available to patients with co- morbidities and reducing the development of resistance to a particular therapy is to use combinations of therapeutic agents.
  • the effects of a combination of therapeutic agents may be additive, synergistic or antagonistic and it is not always predictable whether a combination is likely to be additive, synergistic or antagonistic, or whether reducing therapeutic dosages will be effective.
  • combination therapies that provide patients with benefits such as lower dosages, reduced side effects, synergistic effects, increased therapy options and/or improved treatment success rates.
  • Epoxytiglienone compounds have potent anti-tumour activity. When administered intra- tumourally, epoxytiglienones initiate rapid haemorrhagic necrosis of the tumour mass by directly disrupting tumour vasculature (Boyle et al. 2014).
  • the present disclosure is predicated at least in part on the discovery that the epoxytigliane compound, Compound 1, is able to synergise with cisplatin, 5 fluorouracil, doxorubicin and irradiation in a variety of cancers, including squamous cell carcinomas, adenocarcinomas and fibrosarcomas.
  • a method of treating a tumour comprising administering a combination of an epoxytigliane compound and a second therapeutic agent; wherein the epoxytigliane compound is a compound of formula (I): or a geometric isomer or stereoisomer or a pharmaceutically acceptable salt thereof; wherein R 1 is hydrogen or C 1-6 alkyl; R 2 is -OH or –OR 9 ; R 3 is -OH or –OR 9 ; R 4 and R 5 are independently selected from hydrogen and C 1-6 alkyl; R 6 is hydrogen or –R 10 ; R 7 is hydroxy or OR 10 ; R 8 is hydrogen or C 1-6 alkyl; R 9 is -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl, -C(O)C 1-20 alkyl, -C(O)C 2- 20 alkenyl, -C(O)C 2-20 alkynyl, -C(O)cycloalkyl
  • a combination of epoxytigliane compound and second therapeutic agent for use in the treatment of a tumour wherein the epoxytigliane compound is a compound of formula (I): or a geometric isomer or stereoisomer or a pharmaceutically acceptable salt thereof; wherein R 1 is hydrogen or C 1-6 alkyl; R 2 is -OH or –OR 9 ; R 3 is -OH or –OR 9 ; R 4 and R 5 are independently selected from hydrogen and C 1-6 alkyl; R 6 is hydrogen or –R 10 ; R 7 is hydroxy or OR 10 ; R 8 is hydrogen or C 1-6 alkyl; R 9 is -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl, -C(O)C 1-20 alkyl, -C(O)C 2- 20 alkenyl, -C(O)C 2-20 alkynyl, -C(O)cycloalkyl, -C(O)C 1-
  • an epoxytigliane compound in the manufacture of a first medicament and a second chemotherapeutic agent in the manufacture of a second medicament as a combination therapy for treating a tumour; wherein the epoxytigliane compound is a compound of formula (I):
  • R 1 is hydrogen or C 1-6 alkyl
  • R 2 is -OH or –OR 9
  • R 3 is -OH or –OR 9
  • R 4 and R 5 are independently selected from hydrogen and C 1-6 alkyl
  • R 6 is hydrogen or –R 10
  • R 7 is hydroxy or OR 10
  • R 8 is hydrogen or C 1-6 alkyl
  • R 9 is -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl, -C(O)C 1-20 alkyl, -C(O)C 2- 20 alkenyl, -C(O)C 2-20 alkynyl, -C(O)cycloalkyl, -C(O)C 1- 10 alkylcycloalkyl; -C(O)C 2-10 alkenylcycloalkyl, -C(O)C 2- 10 alkynylcycloalkyl;
  • an epoxytigliane compound in the manufacture of a medicament in a combination therapy with irradiation for treating a tumour; wherein the epoxytigliane compound is a compound of formula (I): or a geometric isomer or stereoisomer or a pharmaceutically acceptable salt thereof; wherein R 1 is hydrogen or C 1-6 alkyl; R 2 is -OH or –OR 9 ; R 3 is -OH or –OR 9 ; R 4 and R 5 are independently selected from hydrogen and C 1-6 alkyl; R 6 is hydrogen or –R 10 ; R 7 is hydroxy or OR 10 ; R 8 is hydrogen or C 1-6 alkyl; R 9 is -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl, -C(O)C 1-20 alkyl, -C(O)C 2- 20 alkenyl, -C(O)C 2-20 alkynyl, -C(O)cyclo
  • Figure 1a is a plot of individual SCC-15 tumour volumes following treatment with combinations of intratumoural Compound 1, intraperitoneal cisplatin and control agents in the BALB/c Foxn1 nu xenogeneic mouse model over time.
  • Figure 1b is a plot of average SCC-15 tumour volumes following treatment with combinations of intratumoural Compound 1, intraperitoneal cisplatin and control agents in the BALB/c Foxn1 nu xenogeneic mouse model over time.
  • Figure 2 is a plot of Kaplan-Meier survival curves for SCC-15 tumours following treatment with combinations of intratumoural Compound 1, intraperitoneal cisplatin and control agents in the BALB/c Foxn1 nu xenogeneic mouse model over time.
  • Figure 3a is a plot of individual UV-13-1 tumour volumes following treatment with combinations of intratumoural Compound 1, intraperitoneal cisplatin and control agents in the C3H/HeNCr syngeneic mouse model over time.
  • Figure 3b is a plot of average UV-13-1 tumour volumes following treatment with combinations of intratumoural Compound 1, intraperitoneal cisplatin and control agents in the C3H/HeNCr syngeneic mouse model over time.
  • Figure 4 is a plot of Kaplan-Meier survival curves for UV-13-1 tumours following treatment with combinations of intratumoural Compound 1, intraperitoneal cisplatin and control agents in the C3H/HeNCr syngeneic mouse model over time.
  • Figure 5a is a plot of individual SCC-15 tumour volumes following treatment with combinations of intratumoural Compound 1, external beam irradiation and control agents in the BALB/c Foxn1 nu xenogeneic mouse model over time.
  • Figure 5b is a plot of average SCC-15 tumour volumes following treatment with combinations of intratumoural Compound 1, external beam irradiation and control agents in the BALB/c Foxn1 nu xenogeneic mouse model over time.
  • Figure 6 is a plot of Kaplan-Meier survival curves for SCC-15 tumours following treatment with combinations of intratumoural Compound 1, external beam irradiation and control agents in the BALB/c Foxn1 nu xenogeneic mouse model over time.
  • Figure 7a is a plot of individual MC38 tumour volumes following treatment with combinations of intratumoural Compound 1, external beam irradiation and control agents in the C57BL/6 syngeneic mouse model over time.
  • Figure 7b is a plot of average MC38 tumour volumes following treatment with combinations of intratumoural Compound 1, external beam irradiation and control agents in the C57BL/6 syngeneic mouse model over time.
  • Figure 8 is a plot of Kaplan-Meier survival curves for MC38 tumours following treatment with combinations of intratumoural Compound 1, intraperitoneal cisplatin and control agents in the C57BL/6 syngeneic mouse model over time.
  • Figure 9a is a plot of cell survival for A431 cells following concurrent treatment with combinations of cisplatin and different concentrations of Compound 1 with each data point representing the mean of three different experiments.
  • Figure 9b is a plot of cell survival for SCC-15 cells following concurrent treatment with combinations of cisplatin and different concentrations of Compound 1 with each data point representing the mean of three different experiments.
  • Figure 10a is a plot of cell survival for A431 cells following concurrent treatment with combinations of 5-fluorouracil and different concentrations of Compound 1 with each data point representing the mean of three different experiments.
  • Figure 10b is a plot of cell survival for SCC-15 cells following concurrent treatment with combinations of 5-fluorouracil and different concentrations of Compound 1 with each data point representing the mean of three different experiments.
  • Figure 11a is a plot of cell survival for A431 cells following concurrent treatment with combinations of Compound 1 and different concentrations of irradiation with each data point representing the mean of three different experiments.
  • Figure 11b is a plot of cell survival for SCC-15 cells following concurrent treatment with combinations of Compound 1 and different concentrations of irradiation with each data point representing the mean of three different experiments.
  • Figure 12 is a Combination-Effect (Fa-CI) plot for A431 cells demonstrating a relationship between Compound 1 and cisplatin, 5-fluorouracil and irradiation.
  • Figure 13 is a Combination-Effect (Fa-CI) plot for SCC-15 cells demonstrating relationship between Compound 1 and cisplatin, 5-fluorouracil and irradiation.
  • Figure 14a is a plot of Kaplan-Meier survival curves for B16-F10-OVA tumours having a tumour size greater than 100 mm 3 following treatment with combinations of intratumoural Compound 1 (5 ⁇ g or 10 ⁇ g) with intraperitoneal doxorubicin, and control agents in C57BL/6 mice over time.
  • Figure 14b is a plot of Kaplan-Meier survival curves for B16-F10-OVA tumours (overall survival) following treatment with combinations of intratumoural Compound 1 (5 ⁇ g or 10 ⁇ g) with intraperitoneal doxorubicin, and control agents in C57BL/6 mice over time.
  • Figure 15a is a plot of Kaplan-Meier survival curves for Lewis Lung Carcinoma (LLC) tumours having a tumour size greater than 100 mm 3 following treatment with combinations of intratumoural Compound 1 (5 ⁇ g or 10 ⁇ g) with intraperitoneal doxorubicin, and control agents in C57BL/6 mice over time.
  • LLC Lewis Lung Carcinoma
  • Figure 15b is a plot of Kaplan-Meier survival curves for Lewis Lung Carcinoma (LLC) tumours (overall survival) following treatment with combinations of intratumoural Compound 1 (5 ⁇ g or 10 ⁇ g) with intraperitoneal doxorubicin, and control agents in C57BL/6 mice over time.
  • LLC Lewis Lung Carcinoma
  • the term “about” refers to a quantity, level, value, dimension, size, or amount that varies by as much as 25%, 20%, 15% or 10% to a reference quantity, level, value, dimension, size, or amount.
  • the term “and/or”, e.g., “X and/or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.
  • the words “comprise”, “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.
  • first Unless otherwise indicated, terms such as “first,” “second,” etc. are used herein merely as labels, and are not intended to impose ordinal, positional, or hierarchical requirements on the items to which these terms refer. Moreover, reference to a “second” item does not require or preclude the existence of lower-numbered item (e.g., a “first” item) and/or a higher-numbered item (e.g., a “third” item). As used herein, the phrase “at least one of”, when used with a list of items, means different combinations of one or more of the listed items may be used and only one of the items in the list may be needed. The item may be a particular object, thing, or category.
  • “at least one of” means any combination of items or number of items may be used from the list, but not all of the items in the list may be required.
  • “at least one of item A, item B, and item C” may mean item A; item A and item B; item B; item A, item B, and item C; or item B and item C.
  • “at least one of item A, item B, and item C” may mean, for example and without limitation, two of item A, one of item B, and ten of item C; four of item B and seven of item C; or some other suitable combination.
  • alkyl refers to optionally substituted linear and branched hydrocarbon groups having 1 to 20 carbon atoms.
  • the alkyl group may have a specified number of carbon atoms, for example, -C 1 -C 6 alkyl which includes alkyl groups having 1, 2, 3, 4, 5 or 6 carbon atoms in linear or branched arrangements.
  • alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, s- and t-butyl, pentyl, 2-methylbutyl, 3-methylbutyl, hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-ethylbutyl, 3-ethylbutyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl and pentadecyl.
  • alkenyl refers to optionally substituted, unsaturated linear or branched hydrocarbons, having 2 to 20 carbon atoms and having at least one double bond. Where appropriate, the alkenyl group may have a specified number of carbon atoms, for example, C 2 -C 6 alkenyl which includes alkenyl groups having 2, 3, 4, 5 or 6 carbon atoms in linear or branched arrangements.
  • alkenyl groups include, ethenyl, propenyl, isopropenyl, butenyl, s- and t-butenyl, pentenyl, hexenyl, hept-1,3-diene, hex-1,3-diene, non-1,3,5-triene and the like.
  • alkynyl refers to optionally substituted unsaturated linear or branched hydrocarbons, having 2 to 20 carbon atoms, having at least one triple bond.
  • the alkynyl group may have a specified number of carbon atoms, for example, C 2 -C 6 alkynyl which includes alkynyl groups having 2, 3, 4, 5 or 6 carbon atoms in linear or branched arrangements.
  • Non-limiting examples include ethynyl, propynyl, butynyl, pentynyl and hexynyl.
  • cycloalkyl and “carbocyclic” refer to optionally substituted saturated or unsaturated mono-cyclic, bicyclic or tricyclic hydrocarbon groups.
  • the cycloalkyl group may have a specified number of carbon atoms, for example, C 3 -C 6 cycloalkyl is a carbocyclic group having 3, 4, 5 or 6 carbon atoms.
  • Non-limiting examples may include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl and the like.
  • “Aryl” means a C 6 -C 14 membered monocyclic, bicyclic or tricyclic carbocyclic ring system having up to 7 atoms in each ring, wherein at least one ring is aromatic.
  • aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl and biphenyl.
  • the aryl may comprise 1-3 benzene rings. If two or more aromatic rings are present, then the rings may be fused together, so that adjacent rings share a common bond.
  • substituents include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, vinyl, methoxy, ethoxy, propoxy, isopropoxy, butoxy, methylthio, ethylthio, propylthio, isopropylthio, butylthio, hydroxy, hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, fluoro, chloro, bromo, iodo, cyano, nitro, -CO 2 H, -CO 2 CH 3 , -C(O)CH 3 , trifluoromethyl, trifluoromethoxy, trifluoromethylthio, difluoromethyl, difluoromethoxy, difluoromethylthio, morpholino, amino, methylamino, dimethylamino, phenyl, phenoxy, phenylcarbon
  • the epoxytigliane compounds may be in the form of pharmaceutically acceptable salts. It will be appreciated however that non-pharmaceutically acceptable salts also fall within the scope of the invention since these may be useful as intermediates in the preparation of pharmaceutically acceptable salts or may be useful during storage or transport.
  • Suitable pharmaceutically acceptable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, maleic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benezenesulphonic, salicyclic sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.
  • pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, ni
  • Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium.
  • Basic nitrogen-containing groups may be quarternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others. It will also be recognised that the epoxytigliane compounds may possess asymmetric centres and are therefore capable of existing in more than one stereoisomeric form.
  • the invention thus also relates to compounds in substantially pure isomeric form at one or more asymmetric centres e.g., greater than about 90% ee, such as about 95% or 97% ee or greater than 99% ee, as well as mixtures, including racemic mixtures, thereof.
  • Such isomers may be obtained by isolation from natural sources, by asymmetric synthesis, for example using chiral intermediates, or by chiral resolution.
  • the compounds of the invention may exist as geometrical isomers.
  • the invention also relates to compounds in substantially pure cis (Z) or trans (E) forms or mixtures thereof.
  • the compounds of the present invention may be obtained by isolation from a plant or plant part, or by derivatisation of the isolated compound, or by derivatisation of a related compound.
  • epoxytigliane compound refers to a generally to a compound having the following basic carbon cyclic structure: The compounds have a tricyclo[9.3.0.0]tetradecane system with a fused cyclopropane ring appended to the six membered ring. The epoxide is fused to the seven membered ring in the 6,7- position.
  • the epoxytigliane compound is generally an epoxytiglien-3-one compound.
  • epoxytiglien-3-one compound refers to a compound having an epoxy-tigliane structure defined above where the five membered ring has a 1,2-ene-3-one structure: .
  • epoxytiglienone compound is used to refer to epoxytiglienone compounds of formula (I).
  • the term “in combination with” as used herein refers to the epoxytigliane compound and the second chemotherapeutic agent (e.g. cancer chemotherapeutic agent) being administered in a single composition, or the second chemotherapeutic agent (e.g. cancer chemotherapeutic agent) or irradiation is administered separately, either simultaneously or sequentially.
  • the epoxytigliane compound and the second chemotherapeutic agent e.g.
  • cancer chemotherapeutic agent or irradiation may be administered at different times and different frequencies but in combination they exert biological effects at the same time or at overlapping times.
  • Methods of Treatment The present invention relates to methods of treating tumours, the method comprising the administration of an epoxytigliane compound or a pharmaceutically acceptable salt thereof in combination with second chemotherapeutic agent or irradiation.
  • the tumour being treated is a tumour to which the epoxytigliane compound may be delivered in a localised way directly to the tumour.
  • the tumour is a cutaneous tumour or subcutaneous tumour or a tumour accessible from the outside of the body, for example, a tumour that is palpable.
  • the tumour is an internal tumour.
  • the localised delivery is achieved during surgery when the tumour is exposed and able to be injected with the epoxytigliane compound.
  • the tumour is internally located and the epoxytigliane compound is delivered by injection guided by an imaging technique, for example, guided by endoscopic ultrasound or by stereotactic imaging.
  • the epoxytigliane compound is delivered to one or more tumours systemically.
  • the tumour is a benign tumour.
  • the tumour is a malignant tumour.
  • the tumour is a primary tumour and in other embodiments, the tumour is a metastatic tumour.
  • the tumour is a cutaneous tumour.
  • cutaneous tumours include seborrheic keratosis, actinic keratosis, basal cell carcinoma (BCC) including nodular BCC, superficial BCC, infiltrative BCC and micronodular BCC, squamous cell carcinoma (SCC), including in-situ SCC and invasive SCC, adenocarcinoma, melanoma including superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral lentginous melanoma and desmoplastic/neutropic melanoma, sarcomas, cutaneous B cell lymphoma and cutaneous T cell lymphoma.
  • BCC basal cell carcinoma
  • SCC squamous cell carcinoma
  • adenocarcinoma melanoma including superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral
  • Examples of cutaneous and subcutaneous tumours that may be treated include angiokeratoma, pyogenic granuloma, cherry angioma, glomus tumour, angiosarcoma, karposi sarcoma, Ewings sarcoma, malignant fibrous histiocytoma, leiomyosarcoma, rhabdomyosarcoma, liposarcoma, synovial sarcoma, stromal sarcoma, gastrointestinal stromal sarcoma, malignant peripheral nerve sheath tumour, primitive neuroectodermal tumour, neurofibroma, Merkel cell carcinoma, dermatofibroma, fibrosarcoma, epithelioid sarcoma and mastocytoma (mast cell tumour).
  • the internal tumour may be any tumour that is accessible to injection during surgery or by guided injection or one that may be treated with systemically administered epoxytigliane and include tumours of the brain, lung, colon, epidermoid, squamous cell, bladder, stomach, pancreas, breast, head, neck, renal system, kidney, liver, ovary, prostate, uterus, oesophagus, testicles, cervix, vagina, thyroid or skin.
  • the tumour is a tumour of the head or neck.
  • the head or neck tumour is a cutaneous tumour.
  • the head or neck tumour is a mucosal epithelial tumour, for example, occurring in the mucosal epithelium of the upper aerodigestive tract.
  • the tumour is a head and neck squamous cell carcinoma (HNSCC).
  • the tumour is a mucosal head or neck cancer, especially a mucosal squamous cell carcinoma (SCC).
  • the mucosal tumour is associated with smoking and/or alcohol use.
  • the tumour is of the oropharynx, especially the tonsils or tongue base.
  • the tumour is associated with human papillomavirus (HPV).
  • the HPV associated tumour is a mucosal SCC.
  • HPV associated mucosal SCCs include tumours of the oropharynx, vulva, vagina and cervix.
  • the tumour is a non-melantotic skin cancer (NMSC).
  • NMSC non-melantotic skin cancer
  • the NMSC may occur on any part of the body.
  • the tumour may be associated with ultraviolet solar radiation exposure, skin phenotype, immunosuppression, xeroderma pigmentosum, chemical exposure, chronic ulcers and scars, ionizing radiation or a combination thereof.
  • the tumour is a high risk tumour having one or more of the following: a size of greater than 4 cm, a depth > 2mm, presence of perineural or lymphovascular invasion, affecting ears or lips, patient immunosuppression and previous radiotherapy.
  • the tumour is a squamous cell tumour (SCC), for example, a thyroid carcinoma, esophageal carcinoma, SCC of the lung, SCC of the penis (that may be associated with HPV) including Bowen’s disease, Erythroplasia of Queyrat and Bowenoid papulosis, prostate cancer, vaginal cancer, cervical cancer and bladder cancer, particularly bladder cancer associated with schistosomiasis.
  • SCC squamous cell tumour
  • the tumour is a sarcoma, for example, an angiosarcoma, karposi sarcoma, Ewings sarcoma, leiomyosarcoma, rhabdomyosarcoma, liposarcoma, synovial sarcoma, stromal sarcoma, gastrointestinal stromal sarcoma, fibrosarcoma or an epithelioid sarcoma.
  • the sarcoma is a UV induced fibrosarcoma.
  • the tumour is an adenocarcinoma.
  • adenocarcinoma examples include esophageal cancers, pancreatic cancer, prostate cancer, cervical cancer, stomach cancer, invasive ductal breast carcinoma, ductal breast carcinoma in situ, invasive lobular breast carcinoma, colon cancer, colorectal cancer, adenocarcinoma of the lung, cholangiocarcinoma (bile duct cancer) and vaginal cancer.
  • Epoxytigliane Compounds The epoxytigliane compound is a 6,7-epoxytigli-1,2-en-3-one compound of formula (I): or a geometric isomer or stereoisomer or a pharmaceutically acceptable salt thereof; wherein R 1 is hydrogen or C 1-6 alkyl; R 2 is -OH or –OR 9 ; R 3 is -OH or –OR 9 ; R 4 and R 5 are independently selected from hydrogen and C 1-6 alkyl; R 6 is hydrogen or –R 10 ; R 7 is hydroxy or -OR 10 ; R 8 is hydrogen or C 1-6 alkyl; R 9 is -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl, -C(O)C 1-20 alkyl, -C(O)C 2-20 alkenyl, -C(O)C 2- 20 alkynyl, -C(O)cycloalkyl, -C(O)C 1-10 alkylcycl
  • the epoxytigliane compound of formula (I) is a compound of formula (II): or a geometric isomer or stereoisomer or a pharmaceutically acceptable salt thereof; where R 6 , R 7 and R 9 are as defined for formula (I).
  • R 1 is –C 1-3 alkyl, especially –CH 3 ;
  • R 2 is -OC(O)C 1-20 alkyl, -OC(O)C 2-20 alkenyl, -OC(O)C 2-20 alkynyl, -OC(O)cycloalkyl, -OC(O)C 1- 10 alkylcycloalkyl; -OC(O)C 2-10 alkenylcycloalkyl, -OC(O)C 2- 10 alkynylcycloalkyl, -OC(O)aryl, -OC(O)C 1-10 alkylaryl, -OC(O)C 2-10 alkenylaryl, -OC(O)C 2- 10 alkynylaryl, -OC(O)C 1-10 alkylC(O)R 11 , -OC(O)C 2-10 alkenylC(O)R 11 , -OC(O)C 2-10 alkenylC(O)R 11 , -OC
  • the epoxytigliane compound of formula (I) is a compound of formula (Ia): or a geometric isomer or stereoisomer or a pharmaceutically acceptable salt thereof; wherein R 1 is hydrogen or C 1-6 alkyl (e.g. R 1 is C 1-3 alkyl, such as CH 3 ); R 2 is -OH or –OR 9 ; R 3 is -OH or –OR 9 ; R 4 and R 5 are independently selected from hydrogen and C 1-6 alkyl (e.g. R 4 and R 5 are independently C 1-3 alkyl, such as CH 3 ); R 6 is hydrogen or –R 10 ; R 7 is hydroxy or -OR 10 ; R 8 is hydrogen or C 1-6 alkyl (e.g.
  • R 8 is C 1-3 alkyl, such as CH 3 );
  • R 9 is -C 1-20 alkyl, -C 2-20 alkenyl, -C 2-20 alkynyl, -C(O)C 1-20 alkyl, -C(O)C 2- 20 alkenyl, -C(O)C 2-20 alkynyl, -C(O)C3-6cycloalkyl, -C(O)C 1-10 alkylC3- 6 cycloalkyl; -C(O)C 2-10 alkenylC 3-6 cycloalkyl, -C(O)C 2-10 alkynylC 3- 6 cycloalkyl, -C(O)aryl, -C(O)C 1-10 alkylaryl, -C(O)C 2-10 alkenylaryl, -C(O)C 2- 10 alkynylaryl, -C(O)C 1-10 alkylC(O)R 11 , -C
  • R 9 is -C(O)C 1-20 alkyl or -C(O)C 2-20 alkenyl, such as tigloyl, 2-methylbutanoyl, hexanoyl, myristoyl, propanoyl or acetyl);
  • R 10 is -C 1-6 alkyl, -C 2-6 alkenyl, -C 2-6 alkynyl, -C(O)C 1-6 alkyl, -C(O)C 2-6 alkenyl, -C(O)C 2- 6 alkynyl, -C(O)aryl, -C(O)C 1-6 alkylaryl, -C(O)C 2-6 alkenylaryl, or -C(O)C 2-6 alkynylaryl (e.g.
  • R 10 is -C(O)C 1-3 alkyl, such as -C(O)CH 3 ); and R 11 is hydrogen, -C 1-10 alkyl, -C 2-10 alkenyl, -C 2-10 alkynyl, C 3-6 cycloalkyl or aryl.
  • the epoxytigliane compound of formula (I) is a compound of formula (Ib): or a geometric isomer or stereoisomer or a pharmaceutically acceptable salt thereof; wherein R 1 is C 1-3 alkyl (e.g.
  • R 1 is CH 3 );
  • R 2 is –OR 9 ;
  • R 3 is –OR 9 ;
  • R 4 and R 5 are independently selected from C 1-3 alkyl;
  • R 6 is hydrogen or –R 10 ;
  • R 7 is hydroxy;
  • R 8 is C 1-3 alkyl;
  • R 9 is -C(O)C 1-20 alkyl or -C(O)C 2-20 alkenyl (e.g. R 9 is tigloyl, 2-methylbutanoyl, hexanoyl, myristoyl, propanoyl or acetyl);
  • R 10 is -C(O)C 1-6 alkyl (e.g.
  • R 10 is -C(O)C 1-3 alkyl, such as -C(O)CH 3 ).
  • the compounds of formulae (I), (II), (Ia) and/or (Ib) have stereochemistry as shown in formula (III) below:
  • the epoxide in the 6,7-position is above the plane of the ring system. In other embodiments, the epoxide in the 6,7-position is below the plane of the ring system.
  • the R 2 group in the 12 position is S and in other embodiments, the R 2 group in the 12 position is R.
  • the epoxytigliane compound of formula (III) is a compound of formula (IIIa): or a pharmaceutically acceptable salt thereof; wherein R 1 is selected from hydrogen and C 1-6 alkyl (e.g.
  • R 1 is C 1-3 alkyl, such as CH 3 );
  • R 2 is selected from -OC(O)C 1-7 alkyl, -OC(O)C 2-7 alkenyl and -OC(O)C 2-7 alkynyl (e.g. R 2 is -O-tigloyl, -O-2-methylbutanoyl, -O-hexanoyl, -O-myristoyl or -O-propanoyl);
  • R 3 is selected from -OC(O)C 1-7 alkyl, -OC(O)C 2-7 alkenyl and -OC(O)C 2-7 alkynyl (e.g.
  • R 3 is -O-tigloyl, -O-2-methylbutanoyl, -O-hexanoyl, or -O-acetyl);
  • R 4 and R 5 are independently selected from hydrogen and C 1-6 alkyl (e.g. R 4 and R 5 are independently C 1-3 alkyl, such as CH 3 );
  • R 6 is selected from hydrogen and -C(O)C 1-6 alkyl (e.g. R 6 is -C(O)C 1-3 alkyl, such as -C(O)CH 3 );
  • R 7 is hydroxy; and
  • R 8 is selected from hydrogen or C 1-6 alkyl (e.g. R 8 is C 1-3 alkyl, such as CH 3 ).
  • the epoxytigliane compound of formula (III) is a compound of formula (IIIb): or a pharmaceutically acceptable salt thereof; wherein R 1 is selected from C 1-6 alkyl (e.g. R 1 is C 1-3 alkyl, such as CH 3 ); R 2 is selected from -OC(O)C 1-7 alkyl and -OC(O)C 2-7 alkenyl (e.g.
  • R 2 is -O-tigloyl, -O-2-methylbutanoyl, -O-hexanoyl, -O-myristoyl or -O-propanoyl);
  • R 3 is selected from -OC(O)C 1-7 alkyl and -OC(O)C 2-7 alkenyl (e.g. R 3 is -O-tigloyl, -O-2-methylbutanoyl, -O-hexanoyl, or -O-acetyl);
  • R 4 and R 5 are independently C 1-6 alkyl (e.g.
  • R 4 and R 5 are independently C 1-3 alkyl, such as CH 3 );
  • R 6 is selected from hydrogen and -C(O)C 1-6 alkyl (e.g. R 6 is hydrogen or -C(O)C 1-3 alkyl, such as -C(O)CH 3 );
  • R 7 is hydroxy;
  • R 8 is selected from C 1-6 alkyl (e.g. R 8 is C 1-3 alkyl, such as CH 3 ).
  • the epoxytigliane compound is selected from: 12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 1); 12,13-di-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 2); 12-hexanoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 3); 12,13-dihexanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 4); 12-myristoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Com
  • the epoxytigliane compound is tigilanol tiglate (abbreviated as “TT”), which is (4S,5S,6R,7S,8R,9R,10S,11R,12R,13S,14R)-12-(2E)-2-methyl-2-enoatyl-13((2S)-2- methylbutyroyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (which is also referred to as [(1R,2S,4R,5S,6S,10S,11R,12R,13R,14S,16R)-5,6,11-trihydroxy-4-(hydroxymethyl)- 8,12,15,15-tetramethyl-13-[(E)-2-methylbut-2-enoyl]oxy-7-oxo-3- oxapentacyclo[9.5.0.0 2,4 .0 6,10 .0 14,16 ]hexadec-8
  • the second therapeutic agent is a chemotherapeutic agent. In other embodiments, the second therapeutic agent is irradiation.
  • the epoxytigliane compounds of formula (I) may synergise with cancer therapies that damage DNA or inhibit tumour-associated host-derived cells that support the growth and/or invasion of tumour cells.
  • the second therapeutic agent is therefore one that damages DNA, resulting in cell death or one that kills tumour- associated host-derived cells, such as myeloid-derived suppressor cells (MDSC), which support the growth and/or invasion of tumour cells.
  • MDSC myeloid-derived suppressor cells
  • agents that damage DNA include DNA intercalating agents (which are substances that insert into the DNA structure of a cell and bind to the DNA to cause DNA damage) and DNA alkylating agents.
  • the second therapeutic agent is a chemotherapeutic agent selected from: i) A platinum chemotherapeutic agent, especially cisplatin and carboplatin, more especially cisplatin (Dasari and Tchounwou, 2013; Bracci et al., 2014); ii) A DNA intercalating agent or inhibitor of the enzyme topoisomerase II, especially doxorubicin or mitoxantrone (Cheung-Ong et al.
  • a DNA alkylating agent such as cyclophosphamide (Cheung-Ong et al. 2013; Hall and Tilby, 1992);
  • An antimetabolite such as gemcitabine or fluorouracil, especially 5-fluorouracil (Cheung-Ong et al.
  • the second therapeutic agent is, or comprises, cisplatin or carboplatin. In some embodiments, the second therapeutic agent is, or comprises, cisplatin.
  • the second therapeutic agent is, or comprises, 5-fluorouracil. In some embodiments, the second therapeutic agent is, or comprises, doxorubicin. Doxorubicin is considered to damage DNA through intercalation or direct alkylation and may result in the formation of DNA double-strand breaks.
  • the second therapeutic agent is irradiation. Irradiation may for example occur by external beam radiation therapy (where high-energy beams are aimed at the site of the cancer from a machine outside the body) or by brachytherapy (where the beams come from a radiation source placed next to or inside the body). At high doses, radiation therapy (or ‘radiotherapy’) kills cancer cells and/or slows their growth by damaging their DNA.
  • the radiation source may be any radioactive source typically used in cancer radiation therapy.
  • the radiation source is caesium (e.g. caesium 137 ).
  • the radiation source is cobalt (e.g. cobalt 60 ).
  • the method of treatment comprises the use of an epoxytigliane compound (e.g.
  • the method of treatment comprises the use of an epoxytigliane compound (e.g.
  • the method of treatment comprises the use of an epoxytigliane compound (e.g.
  • the method of treatment comprises the use of an epoxytigliane compound (e.g.
  • the method of treatment comprises the use of an epoxytigliane compound (e.g.
  • the second chemotherapeutic agent is not an immune checkpoint inhibitor.
  • the epoxytigliane and the second therapeutic agent may be administered in accordance with any suitable dosing regimen. For example, they may be administered simultaneously, sequentially, or on different occasions. In some embodiments, the therapeutic regime involves administration only of the two components, i.e.
  • the epoxytigliane compound and the second therapeutic agent may involve administration of the epoxytigliane compound and a chemotherapeutic agent that damages DNA only.
  • it may involve administration of the epoxytigliane compound and a chemotherapeutic agent that inhibits tumour-associated host-derived cells that support the growth and/or invasion of tumour cells only.
  • the therapeutic regime may involve administration of the epoxytigliane compounds and irradiation only.
  • the therapeutic regime involves administration of the epoxytigliane compound and only one second therapeutic agent, e.g. one chemotherapeutic agent that damages DNA, or one chemotherapeutic agent that inhibits tumour-associated host-derived cells that support the growth and/or invasion of tumour cells.
  • the therapeutic regime involves administration of the epoxytigliane compound and two or more of the second therapeutic agents (for example, two, three, four or five of the second therapeutic agents), e.g. it may involve administration of two chemotherapeutic agents that damage DNA, or two chemotherapeutic agents that inhibit tumour-associated host- derived cells that support the growth and/or invasion of tumour cells, or one of each of those two types of chemotherapeutic agents.
  • the therapeutic regime does not include administration of any therapeutic agents other than the epoxytigliane compound and a second therapeutic agent.
  • the therapeutic regime may involve administration of the epoxytigliane compound, a second therapeutic agent as defined herein, and one or more (e.g.
  • compositions While the epoxytigliane compounds or pharmaceutically acceptable salts thereof and second chemotherapeutic agent, may be administered neat, it may be more convenient to administer the epoxytigliane compounds and second chemotherapeutic agents in the form of one or more pharmaceutical compositions, each together with a pharmaceutically acceptable carrier, diluent and/or excipient. Dosage form and rates for pharmaceutical use and compositions are readily determinable by a person of skill in the art.
  • the epoxytigliane compound is formulated for administration directly onto or into the tumour being treated.
  • the epoxytigliane compound is formulated for topical administration in the form of a gel, ointment, lotion, cream or transdermal patch that may be applied directly onto the tumour being treated.
  • the epoxytigliane compound is formulated for injection, especially intratumoural injection where the compound is injected into one or more places in a tumour.
  • the second chemotherapeutic agent may be administered in any means that is able to deliver the molecule systemically or locally.
  • the second chemotherapeutic agent is conveniently delivered by injection, for example, intravenous, intraarticular, intramuscular, intradermal, subcutaneous or intraperitoneal injection.
  • the second chemotherapeutic agent may also be formulated for local delivery by injection, for example, intratumourally.
  • compositions of the second chemotherapeutic agents may also be incorporated into the compositions of the second chemotherapeutic agents.
  • the epoxytigliane compound and the second chemotherapeutic agent are delivered separately, either simultaneously or sequentially.
  • the epoxytigliane compound and the second chemotherapeutic agent are delivered in a single composition, for example, a single composition suitable for intratumoural delivery or a single composition formulated for systemic delivery.
  • a pharmaceutical composition comprising an epoxytigliane compound or a pharmaceutically acceptable salt thereof and a second chemotherapeutic agent, optionally together with one or more pharmaceutically acceptable carriers.
  • the pharmaceutical composition(s) comprise a pharmaceutically acceptable excipient or an acceptable excipient.
  • pharmaceutically acceptable excipient is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used.
  • carriers well known in the art may be used. These carriers or excipients may be selected from a group including sugars, starches, cellulose and its derivatives, cyclodextrins, malt, gelatine or other gelling agents, polymers, talc, calcium sulphate, vegetable oils, synthetic oils, alcohols and/or polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water.
  • Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water-propylene glycol solutions.
  • parenteral injection liquid preparations can be formulated as solutions in aqueous 1,2-propanediol, dimethylsulfoxide (DMSO), aqueous solutions of gamma cyclodextrin or 2-hydroxypropyl-beta-cyclodextrin, saline solution or polyethylene glycol solution, with or without buffer.
  • DMSO dimethylsulfoxide
  • a preferred range of pH is 3.0-4.5.
  • Suitable buffers buffer the preparation at pH 3.5-4.5 and include, but are not limited to, acetate buffer and citrate buffer.
  • compositions of epoxytigliane compound and/or second chemotherapeutic agent may thus be formulated for parenteral administration (e.g. by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative.
  • the compositions may take such forms as suspensions, solutions, gels or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and/or dispersing agents.
  • the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution, for constitution with a suitable vehicle, e.g.
  • compositions of epoxytigliane compound and/or second chemotherapeutic agent suitable for administration may be presented in discrete units such as syringes, vials, tubes or sachets each containing a predetermined amount of one or more pharmaceutically active compounds or extracts of the invention, as a powder or granules or as a solution or a suspension in an aqueous liquid, a cyclodextrin solution, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil emulsion or as a solution or suspension in a cream or gel or as a suspension of micro- or nano-particles incorporating an epoxytigliane compound, including but not limited to silica or polylactide micro- or nano-particles.
  • compositions may be prepared by any of the method of pharmacy but all methods include the step of bringing into association one or more pharmaceutically active compounds of the invention with the carrier which constitutes one or more necessary ingredients.
  • the compositions are prepared by uniformly and intimately admixing the agents of the invention with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product in to the desired presentation.
  • the epoxytigliane compound and/or second chemotherapeutic agent may be formulated as gels, ointments, emulsions, pastes, creams or lotions, or as a transdermal patch. Gels may be prepared using suitable thickening agents and adding them to aqueous/alcoholic compositions of compound.
  • Suitable thickening or gelling agents are known in the art, such as the polyvinyl carboxy polymer Carbomer 940.
  • Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents.
  • Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilising agents, dispersing agents, suspending agents, thickening agents, or colouring agents.
  • Formulations suitable for topical administration also include solutions or suspensions that may be administered topically in the form of a bath or soak solution or a spray or may be absorbed into a dressing.
  • the second chemotherapeutic agent when it is a small molecule, it may be delivered by any suitable means including oral, topical, rectal, parenteral, sublingual, buccal, intravenous, intraarticular, intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, transdermal and the like as known in the art of pharmacy. Dosage regimens
  • the epoxytigliane compound is delivered in the same composition as the second chemotherapeutic agent.
  • the second chemotherapeutic agent is administered in a separate composition from the epoxytigliane compound. When the second therapeutic agent is irradiation, this is of course, delivered separately from the administration of the epoxytigliane compound.
  • the epoxytigliane compound is administered directly to the tumour, for example by topical administration or by intra-tumoural injection. In some embodiments, the epoxytigliane compound is administered to the tumour once. In other embodiments, the treated tumour is monitored and further administration of epoxytigliane compound may be required if the tumour does not fully respond to the treatment. In embodiments where the tumour is treated topically, the epoxytigliane compound may be administered on a number of occasions over a period of time, for example, daily for a week, or once a week for 4 to 10 weeks. A person skilled in the art, monitoring the subject being treated would be able to determine an appropriate dosage schedule, which may vary depending on the response to the treatment.
  • the epoxytigliane compound is administered once only by intratumoural injection.
  • the amount of epoxytigliane compound delivered is a sub-therapeutic amount.
  • the second chemotherapeutic agent is administered at least once, prior to or simultaneously or sequentially with the epoxytigliane compound.
  • the second chemotherapeutic agent is administered once, either before, concurrently with or after the administration of the epoxytigliane compound.
  • the second chemotherapeutic agent is administered within the 24 hours before, immediately before, concurrently with, immediately after or within 24 hours after the administration of the epoxytigliane compound, especially immediately before, concurrently with and immediately after the administration of the epoxytigliane.
  • immediately before or immediately after is meant that the administration of the second chemotherapeutic agent occurs less than 2 hours before or after the administration of the epoxytigliane compound, especially less than one hour and more especially less than 30 minutes.
  • multiple doses of the second chemotherapeutic agent are administered over a period of time beginning before or together with administration of the epoxytigliane compound and then continuing after administration of the epoxytigliane compound.
  • the second chemotherapeutic agent is administered more than once and on a regular basis before and after administration of the epoxytigliane compound.
  • the second chemotherapeutic agent is administered before administration of the epoxytigliane compound, sequentially or simultaneously with the administration of the epoxytigliane compound and at least once subsequently to administration of the epoxytigliane compound.
  • the second chemotherapeutic agent may be administered 24 hours prior to administration of the epoxytigliane compound, the second chemotherapeutic agent is then administered sequentially or simultaneously with the epoxytigliane compound, either immediately before or immediately after the administration of the epoxytigliane compound, the second chemotherapeutic agent is then administered one or more times over the next one to three months after administration of the epoxytigliane compound, for example, once a week, once every 5 days, once every 4 days, once every 3 days, every 2 days or every day, especially every 1 to 3 days, more especially every 2 days.
  • Subsequent administration of the second chemotherapeutic agent may continue such that 1 to 10 doses of second chemotherapeutic agent are administered after the administration of the epoxytigliane compound, especially 1 to 8 doses, 1 to 6 doses, 1 to 4 doses, 1 to 3 doses or 1 to 2 doses.
  • the epoxytigliane compound is administered in an effective amount.
  • An "effective amount” means an amount necessary at least partly to attain the desired response, for example, to reduce the size of the tumour or to destroy the tumour in total. The amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the formulation of the composition, the size of the tumour, the assessment of the medical situation, the second therapeutic agent it is combined with and other relevant factors.
  • An effective amount may lie in the range of about 0.1 ⁇ g per cm 3 tumour volume to 20 mg per cm 3 tumour volume per dosage.
  • the dosage is preferably in the range of 1 ⁇ g to 10 mg per cm 3 tumour volume per dosage, such as is in the range of 0.5 ⁇ g to 5 mg per cm 3 tumour volume per dosage.
  • the dosage is in the range of 0.01 to 1 mg per cm 3 tumour volume, for example 0.1 mg to 1 mg per cm 3 tumour volume to 0.7 mg per cm 3 tumour volume, 0.1 to 0.8 mg per cm 3 tumour volume, such as 0.125 mg to 0.7 mg per cm 3 tumour volume.
  • the dosage is in the range of 0.001 mg to 20 mg per dosage, for example, 0.005 mg to 15 mg per dosage, especially 0.05 to 10 mg per dosage, more especially about 0.1 to about 5 mg per dosage. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, in some embodiments, where administration is intra-tumoural, the epoxytigliane compound is administered once and the progress of treatment monitored. In some embodiments, if the tumour does not completely resolve or if the tumour recurs, a second dose may be administered. In some embodiments, where the administration is topical, the topical compound formulation may be administered directly onto the site of the tumour in the form of a gel, cream, ointment or lotion.
  • the frequency of treatment will depend on the tumour, its size, the subject being treated and the like.
  • a topical formulation may be applied weekly until the tumour is resolved.
  • the treatment may be a single treatment and a second treatment only administered if the tumour is not completely resolved.
  • the epoxytigliane compound is administered in a sub-therapeutic amount.
  • a sub-therapeutic amount is an amount that is less than an amount expected to be effective if the epoxytigliane compound was administered alone.
  • the sub-therapeutic amount may be at the lower end of the effective amount described above for example, 0.1 to 0.2 mg per cm 3 tumour volume.
  • the second chemotherapeutic agent may also be administered in an effective amount.
  • the amount of second chemotherapeutic agent considered to be effective will depend on the subject being treated, their health and physical condition, the identity of the second chemotherapeutic agent, the formulation of the composition and the assessment of the medical situation. It is expected that the amount of the second chemotherapeutic agent will fall within a fairly broad range of amounts.
  • An effective amount may lie in the range of about 0.1 ng per kg to about 500 mg per kg body weight, 100 ⁇ g per kg to 100 mg per kg body weight, 1 mg per kg to 50 mg per kg body weight, 1 mg per kg to 20 mg per kg body weight.
  • the actual dosages may be in the range of from 1 ⁇ g to 1 g, for example, 100 ⁇ g to 750 mg per dose.
  • the effective amount may be a sub-therapeutic amount when used as a monotherapy but when in combination with the epoxytigliane compound is an effective amount.
  • the radiation may be administered at least once. In some embodiments, the radiation is administered just once either within 24 hours before, immediately before, immediately after or within 24 hours after administration of the epoxytigliane compound, especially immediately before or immediately after administration of the epoxytigliane compound.
  • immediately before or immediately after is meant that the irradiation occurs less than 2 hours before or after the administration of the epoxytigliane compound, especially less than one hour and more especially less than 30 minutes.
  • the radiation is administered more than once, for example, within the irradiation may be administered 24 hours prior to administration of the epoxytigliane compound, then administered sequentially or simultaneously with the epoxytigliane compound, either immediately before or immediately after the administration of the epoxytigliane compound, then administered one or more times over the next month after administration of the epoxytigliane compound, for example, once a week, once every 5 days, once every 4 days, once every 3 days, every 2 days or every day.
  • the amount of irradiation administered is between 1 and 8 Gray (Gy) per dose.
  • the amount of irradiation administered is between 1 and 6 Gy, 2 and 4 Gy or about 2 Gy per dose, for example to deliver a total dose between 8 and 50 Gy.
  • irradiation is administered once at a sub-optimal dose, for example, between 2 and 8 Gy total dose, especially 2 to 4 Gy or 1 to 2 Gy.
  • a sub-optimal dose for example, between 2 and 8 Gy total dose, especially 2 to 4 Gy or 1 to 2 Gy.
  • the subject that may be treated with the combination therapy is a mammal, a bird, an aquatic animal such as a fish, or a reptile.
  • the subject is a human, a laboratory animal such as a mouse, rat or rabbit, a companion animal such as a dog or cat, a working animal such as a horse, donkey and the like, a livestock animal such as a cow, bull, pig, sheep, goat, deer, llama, alpaca and the like, or a captive wild animal such as those in zoos or wildlife parks including lions, leopards, cheetah, elephant, zebra, antelope, giraffe, koala, kangaroo and reptiles such as crocodiles, lizards, snakes and the like, a bird, especially a captive bird, such as a budgerigar or canary, cockatoo, parakeet, macaw, parrot and the like, or a fish, especially a captive fish such as tropical fish (zebra fish, guppy, Siamese fighting fish, clown fish, cardinal tetra
  • the subject is a human or a companion animal.
  • the subject is immunocompetent.
  • the subject is immunosuppressed or immunocompromised.
  • Kits The compositions of epoxytigliane compound and the second chemotherapeutic agent may be formulated separately and sold together in a kit or package. Each kit may comprise dosages of each compound to achieve treatment of a tumour.
  • the epoxytigliane composition is formulated for topical administration, such as in a gel, lotion, cream or ointment or is impregnated into a dressing.
  • the epoxytigliane compound is formulated for injection such as intratumoural injection.
  • the epoxytigliane formulation may be present in the kit as a liquid ready for uptake into a syringe, as a powder or solid formulation which may be solubilized in a carrier before injection or may be present in the kit in a pre-filled syringe.
  • Each kit may comprise one or more doses of epoxytigliane compound.
  • the kit will contain a single dose of epoxytigliane compound in a formulation suitable for intratumoural injection.
  • the kit will contain a topical formulation of epoxytigliane compound containing multiple doses for application to the tumour.
  • the second chemotherapeutic agent is formulated for parenteral administration in a single bolus dose or in a multiple dose form.
  • the kit may contain the second chemotherapeutic agent in a pre-filled syringe, as a liquid in a vial ready for uptake into a syringe, or as a solid ready for dissolution before uptake into a syringe.
  • the liquid or solid formulations may be single dose formulations or multiple dose formulations.
  • the kit may contain multiple doses of second chemotherapeutic agent, each formulated separately in a prefilled syringe, as a liquid in a vial ready for uptake into a syringe or as a solid ready for dissolution and uptake into a syringe.
  • the kit may further comprise an insert with instruction for use of each formulation, including how to prepare each dose if required, how to administer each dosage and when to administer each dosage.
  • the kit comprises an epoxytigliane compound (e.g. (4S,5S,6R,7S,8R,9R,10S,11R,12R,13S,14R)-12-(2E)-2-methyl-2-enoatyl-13((2S)-2- methylbutyroyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one), which may for example be for use with irradiation to treat tumours such as squamous cell carcinoma (SCC), including in-situ SCC and invasive SCC, and adenocarcinomas, including colonic adenocarcinomas.
  • SCC squamous cell carcinoma
  • adenocarcinomas including colonic adenocarcinomas.
  • the kit comprises an epoxytigliane compound (e.g. (4S,5S,6R,7S,8R,9R,10S,11R,12R,13S,14R)-12-(2E)-2-methyl-2-enoatyl-13((2S)-2- methylbutyroyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one) and cisplatin.
  • This kit may for example be used to treat squamous cell carcinoma (SCC), including in-situ SCC and invasive SCC.
  • the kit comprises an epoxytigliane compound (e.g.
  • kits may for example be used to treat squamous cell carcinoma (SCC), including in-situ SCC and invasive SCC.
  • the kit comprises an epoxytigliane compound (e.g.
  • mice All mice were house in pathogen-free conditions in the QIMR Animal Facility where they were caged with access to filtered air, food and water were freely available. Mice were handled and procedures performed using aseptic techniques within a laminar flow hood. Prior to inoculation of subject mice, the cancer cells were seeded to T175 tissue culture flasks (Thermo Scientific) to increase cell numbers. Once 70-80% confluent, cells were trypsinized and resuspended in RPMI-1640 media for harvesting and cell count using a haemocytometer. The cell volume was centrifuged and the pellet reconstituted in RPMI-1640 media to a volume sufficient for the number of inoculations required at 100 ⁇ L per injection with two sites per mouse subject.
  • T175 tissue culture flasks Thermo Scientific
  • the cell suspension was kept on ice and transferred to the animal facility for injection into the subjects.
  • the details of the mice used and the cell lines used for the combination experiment is provided in Table 1.
  • the BALB/c Foxn1 nu mice were obtained from the Animal Resource Centre, Western Australia.
  • the C3H/HeNCr mice were bred at the QIMR Animal Facility.
  • Table 1 C3H/HeNCr mice were shaved (Wahl, Illinois, USA) one day before inoculation. Whilst restrained subjects were inoculated on the hind quarter bilaterally by injecting 100 ⁇ L of the appropriate cell suspension into the subcutaneous space with a Terumo® (Tokyo, Japan) 27G x 1 ⁇ 2 “ needle. Subjects were ear tagged for identification.
  • mice were monitored twice weekly for tumour development and well-being and once tumours developed, the subjects were monitored daily.
  • a cohort of thirty BALB/c Foxn1 nu and twenty-one C3H/HeNCr mice, each with two tumours per mouse were divided into six experimental groups as set out in Table 2.
  • 30 ⁇ g Compound 1 is considered a therapeutic dose for a 100 mm 3 tumour so 5 ⁇ g and 10 ⁇ g were selected to be sub-optimal doses.
  • Table 2 Weight was recorded for each subject prior to and post-treatment, and at the time of culling.
  • each tumour was measured using 150 mm digital Vernier callipers (Kinchrome, Qld, AU) and individual tumour volume (mm 3 ) calculated using the formula: (length x (width 2 )) ⁇ 2 Twenty-four hours before it was anticipated that tumours would reach a volume of 70-100 mm 3 , the test subjects were administered either 2.5 mg/kg cisplatin (0.5 mg/mL in 0.9% normal saline) or an equivalent volume of 0.9% normal saline intraperitoneally using a Terumo® 0.5 mL U-100 insulin 29 G x 1 ⁇ 2 “ needle.
  • IP injections were performed by first sterilizing the anterior abdominal wall of the subject with an alcohol swab before the dorsal skin of the subject was scruffed and the subject inverted with head down. A 29 G needle with the bevel upwards is used to inject into the lower right abdominal quadrant. On reaching a volume of 70-100 mm 3 , tumours of the test subjects were injected IT with 50 ⁇ L of either 5 ⁇ g or 10 ⁇ g of Compound 1 or for control subjects 50 ⁇ L of 40% propylene glycol (PG). Test subjects received further IP injections of cisplatin and 0.9% normal saline at day 7 and day 14 following initial injections. Compound 1 concentration was confirmed by high performance liquid chromatography (HPLC) prior to use.
  • HPLC high performance liquid chromatography
  • Test subjects were observed daily and monitored for up to three months following treatment. Tumour volume was recorded as above. The subjects were euthanized by CO 2 inhalation when total tumour burden reached 1000 mm 3 per mouse, if ill, and on completion of the experiment. Data was analysed with Microsoft Excel and Prism 7 to calculate average tumour volumes and standard deviations (SD). Individual and averaged tumour volumes and Kaplan-Meier survival curves were graphed relative to time of treatment.
  • Xenogeneic model Individual SCC-15 tumour volumes and average tumour volumes for the combination of Compound 1 and cisplatin in the BALB/c Foxn1 nu mouse model are shown in Figures 1a and 1b respectively. Kaplan-Meier survival curves for the xenogeneic model are shown in Figure 2.
  • the BALB/c Foxn1 nu mouse model is an immunocompromised host that lacks an intact acquired immune system. This model allows assessment of the Compound 1/cisplatin combination therapy where the host immune system is less likely to be contributing to any treatment effect. From Figures 1 a/b and 2, there is a clear trend across individual and average tumour volumes, and the Kaplan-Meier survival curves that combination therapy has superior efficacy, particularly at the higher concentration of Compound 1 – 2.5 mg/kg cisplatin IP and 10 ⁇ g Compound 1 IT. This combination resulted in a long-lasting response with one tumour remaining cured at 60 days. Lower dose (5 ⁇ g) of Compound 1 was not as effective as the higher dose but still demonstrated superior efficacy to that of single agent therapy or no treatment.
  • Subject weight (g) served as a marker for systemic toxicity of IP cisplatin. This allowed assessment of whether combination therapy with sub-therapeutic concentrations of IP cisplatin demonstrate efficacy whilst avoiding the usual side effects associated with its administration. The results are shown in Table 3. Table 3 The results show that there is very little weight difference pre- and post-treatment, but by the end of the experiment there is a clear weight gain for both the control and cisplatin treated subjects. Although a small weight loss was observed post-treatment with cisplatin, the weight was recovered by the end of the experiment.
  • QIMR Queensland Institute of Medical Research
  • mice All mice were housed in pathogen-free conditions in the QIMR Animal Facility where they were caged with access to filtered air, food and water were freely available. Mice were handled and procedures performed using aseptic techniques within a laminar flow hood. Prior to inoculation of subject mice, the cancer cells were seeded to T175 tissue culture flasks (Thermo Scientific) to increase cell numbers. Once 70-80% confluent, cells were trypsinized and resuspended in RPMI-1640 media for harvesting and cell count using a haemocytometer. The cell volume was centrifuged and the pellet reconstituted in RPMI-1640 media to a volume sufficient for the number of inoculations required at 100 ⁇ L per injection with two sites per mouse subject.
  • T175 tissue culture flasks Thermo Scientific
  • the cell suspension was kept on ice and transferred to the animal facility for injection into the subjects.
  • the details of the mice used and the cell lines used for the combination experiment is provided in Table 6.
  • the BALB/c Foxn1 nu and C57BL/6 mice were obtained from the Animal Resource Centre, Western Australia.
  • Table 6 The MC38 cell line is a murine colonic adenocarcinoma cell line derived from the C57BL/6 mouse (Kerafast, ENH204). C57BL/6 mice were shaved (Wahl, Illinois, USA) one day before inoculation.
  • tumours of the test subjects were irradiated with 0, 5 or 10 Gy. Adequate positioning was ensured with the irradiator (Gammacel 40 Extractor) by sedating each mouse with a combination solution of Ketamine (80 mg/kg of 8 mg/mL solution, Ceva, NSW, AU) and Midazolam (4 mg/kg of 0.4 mg/mL solution, Sandoz, Hozmaschinen, Germany) via intraperitoneal injection.
  • Ketamine 80 mg/kg of 8 mg/mL solution, Ceva, NSW, AU
  • Midazolam 4 mg/kg of 0.4 mg/mL solution, Sandoz, Hozmaschinen, Germany
  • mice were double contained during transfer to and from the irradiator. During irradiation, mice were restrained with lead shielding to prevent exposure to sites other than the flanks where the tumours were located. Immediately upon return to the Animal Facility, mice received intraturmoral (IT) injections with 50 ⁇ L of 7.5 ⁇ g of Compound 1 or for control subjects 50 ⁇ L of 40% propylene glycol (PG) using a Terumo 0.5 mL U-100 insulin 29G x 1 ⁇ 2 “ needle. On completion of treatment, mice were warmed with a heating blanket, placed on their side, and observed until ambulant. Test subjects were observed daily and monitored for up to three months following treatment. Tumour volume was recorded as above.
  • IT intraturmoral
  • PG propylene glycol
  • the subjects were euthanized by CO 2 inhalation when total tumour burden reached 1000 mm 3 per mouse, if ill, and on completion of the experiment. Data was analysed with Microsoft Excel and Prism 7 to calculate average tumour volumes and standard deviations (SD). Individual and averaged tumour volumes and Kaplan-Meier survival curves were graphed relative to time of treatment.
  • Xenogeneic model Individual SCC-15 tumour volumes and average tumour volumes for the combination of Compound 1 and irradiation in the BALB/c Foxn1 nu mouse model are shown in Figures 5a and 5b respectively. Kaplan-Meier survival curves for the xenogeneic model are shown in Figure 6.
  • the BALB/c Foxn1 nu mouse model is an immunocompromised host that lacks an intact acquired immune system.
  • Compound 1 with irradiation either 5 Gy or 10 Gy, improved duration of treatment response at least 4.6 ⁇ for the xenogeneic model (11 vs 51.5 and 111 days) and 8.7 ⁇ for the syngeneic model (6.2 vs 64.6 and 65.9 days). Median survival with each treatment is shown in Table 8.
  • Table 8 Example 3: In vitro experiments with Compound 1 and chemoirradiation The cell lines were investigated in in vitro experiments for assessment of combinations of Compound 1 with cisplatin, 5-fluorouracil and irradiation. The cell lines are given in Table 9 and include one cutaneous cell line and one oropharyngeal mucosal cell line.
  • the cell lines were authenticated by short tandem repeat (STR) profiling and mycoplasma testing as performed by the QIMR Analytical Facility. The number of cells removed during passage was determined using a haemocytometer (Fuchs Rosenthal Dark Lines; Hirschmann Laboratories, Eberstadt, Germany). Cells were seeded at 3000 cells/well in 100 ⁇ L of medium in flat bottom 96 well cell culture plates (Corning Incorporated, New York, USA). Cells were incubated for 24 hr to ensure plate adherence prior to treatment.
  • STR short tandem repeat
  • Each cell line was exposed to a range of dosages of Compound 1 (20 ⁇ g/mL in 100% ethanol), cisplatin (50 ⁇ M stock in 0.9% normal saline, Sigma Aldrich, Missouri, USA), 5-fluorouracil (5- FU, 50 ⁇ M stock in dimethylsulfoxide (DMSO, Sigma Aldrich), and irradiation.
  • Compound 1 concentration was confirmed by HPLC prior to use. Dose ranges are provided in Table 10.
  • Table 10 For each of Compound 1, cisplatin and 5-FU, media from the superior most row of the 96-well plate was aspirated prior to administration of 200 ⁇ L of respective drug stock in RPMI-1640 media formulated to the maximal desired concentration.
  • Combination exposure The cell lines were exposed to combination of Compound 1 and other agent across a range of dosages as shown in Table 11. The dosages were those that resulted in suboptimal cell death ( ⁇ IC 50 ) as evidenced by single agent experimentation.
  • Table 11 Combination experiments between Compound 1 and cisplatin or 5-FU were conducted using varying administration time-courses. To assess the influence of administration time course on treatment efficacy, each cell line was exposed to Compound 124 hours before, concurrently and 24 hours after treatment with either cisplatin or 5-FU. Cell lines treated with a combination of Compound 1 and irradiation were first treated with the allocated duration of irradiation before immediate treatment with Compound 1. For single agent experiments, wells included no treatment and ethanol treatment controls.
  • Sulforhodamine B proliferation assay Following incubation of treated cells for 5-7 days, cell survival was determined using a sulforhodamine B (SRB) proliferation assay. This assay quantitatively assesses the degree of protein staining as a surrogate marker for cellular growth. Cells were washed with PBS and subsequently fixed with methylated spirits (Chem Supply, South Australia) for at least five minutes. Following fixation, the methylated spirits was removed and cells again washed gently with water prior to staining with 50 ⁇ L/well of 0.4% SRB solution (Sigma Aldrich) in 1% acetic acid for sixty minutes.
  • SRB sulforhodamine B proliferation assay. This assay quantitatively assesses the degree of protein staining as a surrogate marker for cellular growth. Cells were washed with PBS and subsequently fixed with methylated spirits (Chem Supply, South Australia) for at least five minutes. Following fixation, the methylated spirits was removed and cells again washe
  • CI Chou-Talalay combination indices
  • Chou (2010) Means, standard deviations and Chou-Talalay combination indices (CI) (Chou (2010)) were determined and used to detect any significant difference between controls and test combination as well as to assess the relationship between Compound 1 and the chemoradiation.
  • the Chou-Talalay combination theorem is a statistical method by which the effect of several agents when used in combination can be quantified by producing a number relative to 1.
  • Combination-effect (CI-Fa) plots depict the combination indices for a range of drug concentrations graphically. Data points that fall below the line (the line represents a combination index of 1) suggest a synergistic relationship. A benefit of the CI-Fa plot is that it demonstrates a trend across a range of effect levels.
  • the x axis or effect (Fa) correlates directly with cell survival.
  • an effect (Fa) of 0.1 is consistent with a cell survival of 10%.
  • Analysis was performed using the software program Combusyn. Results Single Agents Using a 4 hour exposure time, cisplatin alone had an IC 50 between 3.125 and 6.25 ⁇ M in both cell lines. SCC-15 was closer to the lower end, whereas A431 was slightly more resistant and had an IC 50 closer to the upper end. Using a 4 hour exposure time, Compound 1 alone had an IC 50 between 62.5 and 200 ⁇ M, with SCC-15 closer to the lower end and A431 being slightly more resistant and closer to the upper end.
  • the cell survival for the combination of Compound 1 and 5-FU in A431 cells is shown in Figure 10a. It is evident that addition of Compound 1 to any concentration of 5-FU results in a significant increase in cell death compared to 5-FU alone. The approximate IC 50 for the combination therapy was equivalent to or less than 1.56 ⁇ M.
  • the cell survival for the combination of Compound 1 and 5-FU in SCC-15 cells is shown in Figure 10b. It is evident that addition of Compound 1 to any concentration of 5-FU results in a significant increase in cell death compared to cisplatin alone. The approximate IC 50 for the combination therapy was equivalent to or less than 12.5 ⁇ M.
  • A431 cells and SCC-15 cells demonstrated a similar response with increases in both 5-FU and Compound 1 resulting in decreased cell survival.
  • the cell survival for the combination of Compound 1 and irradiation in A431 cells is shown in Figure 11a. It is evident that addition of Compound 1 to any concentration of irradiation results in a significant increase in cell death compared to Compound 1 alone.
  • the cell survival for the combination of Compound 1 and irradiation in SCC-15 cells is shown in Figure 11b. Exposure to Compound 1 and irradiation results in a pronounced fall in cell survival. The combination is superior to that of Compound 1 alone. Increasing irradiation exposure does not greatly improve efficacy.
  • the IC 50 for the combination of Compound 1 and irradiation was 0 to 6.26 ⁇ M for A431 cells and 0 to 1.56 ⁇ M for SCC-15 cells.
  • Combination Effect For a combination therapy with Compound 1 and 5-FU, there was little difference between administration of Compound 1 24 hours before or 24 hours after 5-FU or concurrent administration in SCC-15 cells.
  • Combination Effect The Combination Effect plot for A431 cells is shown in Figure 12 and the combination indices for the IC 50 s for each concentration of Compound 1 is shown in Table 12.
  • Table 12 Using the Chou-Talalay combination index method, these results suggest that in A431 cells, Compound 1 has a synergistic relationship with cisplatin, 5-FU and irradiation. All data points for the combinations have indices ⁇ 1.
  • the Combination Effect plot for SCC-15 cells is shown in Figure 13 and the combination indices for the IC 50 s for each concentration of Compound 1 is shown in Table 13.
  • Example 4 Assessment of efficacy of combined chemotherapy treatment with Compound 1 in mouse models of cancer This example assessed the impact of the chemotherapeutic agent, doxorubicin, in combination with Compound 1 in the treatment on mouse models of cancers, specifically melanoma and lung cancer.
  • mice All animal procedures were approved in accordance with NHMRC guidelines (Australian Code for the Care and Use of Animals for Scientific Purposes 8 th Edition, 2013; National Health and Medical Research Council of Australia) by the QIMR Berghofer Animal Ethics Committee, approval number A2109-621. All mice were housed in a specific pathogen free (SPF) facility, with 12 hours light/dark cycle and continual access to food and water.
  • SPF pathogen free
  • Doxorubicin combination treatment C57BL/6 mice (6-7 weeks old) were injected subcutaneously (s.c.) on both flanks with B16-F10- OVA mouse melanoma cells (2 ⁇ 10 5 cells per site in 50 ⁇ l) or LLC mouse lung cancer cells (5 ⁇ 10 5 cells per site in 50 ⁇ l).
  • Tumours were allowed to develop to approximately 75-100 mm 3 . Mice were then treated intra-peritoneally with doxorubicin in saline (3 mg/kg; 60 ⁇ g/dose, saline for vehicle). The following day, tumours were injected intratumourally with either vehicle (40% PG/30 mM acetate) or 5 or 10 ⁇ g Compound 1 (50 ⁇ l of 100 ⁇ g/ml [5 ⁇ g] or 50 ⁇ l of 200 ⁇ g/ml [10 ⁇ g]) in the 40% PG/30 mM acetate pH 4.30 as previously published (Cullen et al. 2021). Treatment with the chemotherapy agent was repeated after 7 and 14 days.
  • mice per group 10 tumours
  • LLC tumour model 5 mice per group were used in a single experiment (10 tumours).
  • Mice were also assessed for clinical signs of distress according to an approved clinical score sheet during the experiment. The experiment ceased when an unacceptable clinical score (>6) was reached, or the cumulative tumour burden of the mouse exceeded 1,000 mm 3 .
  • doxorubicin treatment in combination with 10 ⁇ g Compound 1 had an effect on tumour growth, with extending time until experimental endpoint when compared to doxorubicin alone and to a lesser extent 10 ⁇ g Compound 1 combined with saline vehicle.
  • a total of 7/20 tumours were completely ablated in the doxorubicin / 10 ⁇ g Compound 1 cohort compared to 4/20 tumours in the saline / 10 ⁇ g Compound 1 group.

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Abstract

L'invention concerne des polythérapies faisant appel à un composé époxytigliane (par exemple, un tiglate de tigilanol) et soit à une irradiation soit à un agent chimiothérapeutique qui est i) un agent chimiothérapeutique endommageant l'ADN (par exemple, le cisplatine), ou ii) un agent chimiothérapeutique inhibant des cellules dérivées d'hôte associées à une tumeur qui supportent la croissance et/ou l'invasion de cellules tumorales (par exemple, le 5-fluoruracile, ou la doxorubicine). L'invention concerne également des compositions pharmaceutiques et des kits contenant des composés époxytiglianes et les agents chimiothérapeutiques.
PCT/AU2023/050108 2022-02-17 2023-02-17 Polythérapies WO2023154984A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018018097A1 (fr) * 2016-07-28 2018-02-01 Qbiotics Limited Méthode de traitement de tumeurs.
WO2018170559A1 (fr) * 2017-03-23 2018-09-27 Qbiotics Limited Traitement d'association pour le traitement ou la prévention de tumeurs

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018018097A1 (fr) * 2016-07-28 2018-02-01 Qbiotics Limited Méthode de traitement de tumeurs.
WO2018170559A1 (fr) * 2017-03-23 2018-09-27 Qbiotics Limited Traitement d'association pour le traitement ou la prévention de tumeurs

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BARNETT, C. M. E. ET AL.: "Optimising intratumoral treatment of head and neck squamous cell carcinoma models with the diterpene ester Tigilanol tiglate", INVESTIGATIONAL NEW DRUGS, vol. 37, no. 1, 2019, pages 1 - 8, XP036722436, DOI: 10.1007/s10637-018-0604-y *
BOYLE, G. M. ET AL.: "Intra-Lesional Injection of the Novel PKC Activator EBC-46 Rapidly Ablates Tumors in Mouse Models", PLOS ONE, vol. 9, no. 10, 2014, pages e108887, XP055460453, DOI: 10.1371/journal.pone.0108887 *
CULLEN JASON K. ET AL: "Activation of PKC supports the anticancer activity of tigilanol tiglate and related epoxytiglianes", SCIENTIFIC REPORTS, vol. 11, no. 207, pages 1 - 14, XP093087420, DOI: 10.1038/s41598-020-80397-9 *
STELFONTA ®: "Virbac, product insert", INTERNET CITATION, 18 October 2021 (2021-10-18), pages 1 - 2, XP093087424, Retrieved from the Internet <URL:https://vet-us.virbac.com/files/live/sites/virbac-b2b-usa/files/client%20leaflet/Products/Stelfonta/STELFONTA%20PI%20Letter%20size%20-FINAL.pdf> [retrieved on 20230929] *

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